DNA polymerases synthesize the formation of DNA molecules which are complementary to a DNA template. Upon binding to a primer terminus of a DNA template, polymerases synthesize DNA in the 5' to 3' direction, successively adding nucleotides to the 3'-hydroxyl group of the growing strand. Thus, in the presence of deoxyribonucleoside triphosphates (dNTPs) and a primed template, a new DNA molecule, complementary to the single stranded DNA template, can by synthesized.
A number of DNA polymerases have been isolated from mesophilic microorganisms such as E. coli. A number of these mesophilic DNA polymerases have also been cloned. Lin et al. cloned and expressed T4 DNA polymerase in E. coli (Proc. Natl. Acad. Sci. USA 84:7000-7004 (1987)). Tabor et al. (U.S. Pat. No. 4,795,699) describes a cloned T7 DNA polymerase, while Minkley et al. (J. Biol. Chem. 259 (16):10386-10392 (1984)) and Chatterjee (U.S. Pat. No. 5,047,342) described cloning of E. coli DNA polymerase I and T5 DNA polymerase, respectively.
Although DNA polymerases from thermophiles are known, relatively little investigation has been done to isolate and clone these enzymes. Chien et al., J. Bacteriol. 127:1550-1557 (1976) describe a purification scheme for obtaining a polymerase from Therrnus aquaticus. The resulting protein had a molecular weight of about 63,000 daltons by gel filtration analysis and 68,000 daltons by sucrose gradient centrifugation. Kaledin et al., Biokhymiya 45:644-651 (1980) disclosed a purification procedure for isolating DNA polymerase from T. aquaticus YT1 strain. The purified enzyme was reported to be a 62,000 dalton monomeric protein. Gelland et al. (U.S. Pat. No. 4,889,818) cloned a gene encoding a thermostable DNA polymerase from Thermus aquaticus. The molecular weight of this protein was found to be about 86,000 to 90,000 daltons.
Thermophilic bacteria, other than Thermus aquaticus, have been isolated and a number of thermostable enzymes have been isolated from these organisms. Bragger et al., Appl. Microbiol. Biotechnol. 31:556-561 (1989) screened thirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities.
DNA polymerases have been isolated from thermophilic bacteria including Thermotoga (Simpson et al., Biochem. Cell. Biol. 86:1292-1296 (1990)); Bacillus stearothermophilus (Stenesh et al., Biochim. Biochys. Acta 272:156-166 (1972); and Kaboev et al., J. Bacteriol. 145:21-26 (1981)); and several archaebacterial species (Rossi et al., System. Appl. Microbiol. 7:337-341 (1986); Klimczak et al., Biochemistry 25:4850-4855 (1986); and Elie et al., Eur. J. Biochem. 178:619-626 (1989)). Innis et al., In PCR Protocol: A Guide To Methods and Amplification, Academic Press, Inc., San Diego (1990) noted that there are several extreme thermophilic eubacteria and archaebacteria that are capable of growth at very high temperatures (Bergquist et al., Biotech. Genet. Eng. Rev. 5:199-244 (1987); and Kelly et al., Biotechnol. Prog. 4:47-62 (1988)) and suggested that these organisms may contain very thermostable DNA polymerases.